(132a) Hydrogels As Scaffolds for Extracellular Vesicle-Based Therapeutics

One of the major challenges in regenerative medicine remains, to date, the repair of critical size bone defects, particularly through underlying conditions, such as post-menopausal osteoporosis. Bone marrow derived mesenchymal stromal cells (MSC) have been used in a variety of clinical trials to promote bone regeneration, due to their elevated secretion of regenerative factors (growth factors, chemokines, extracellular vesicles (EVs)). Despite promising pre-clinical results, the direct delivery of MSCs in vivo leads to low survival and rapid clearance due to acute inflammation, (i.e., <5% of MSCs survive 24 hours post-transplantation), which has limited successful translation to new clinical therapies. EVs are nanosized membranous particles that mediate the complex transmission of molecular signals by delivering biomolecular cargo between cells. Their therapeutic potential lies in the ability to propagate signal transduction between cells and tissues independently of the presence of parent cells. In this study we use our versatile microgel scaffolds to initially answer fundamental questions about the mechanisms of EV endocytosis and subsequently utilize Strain-Promoted Azide-Alkyne Click Chemistry reaction (SPAAC) to engineer a granular biodegradable scaffold for the controlled release of azide-modified anti-inflammatory EVs in rat critical-sized calvarial defect.

Specifically, in the first part of this study, we fabricate 2D hydrogels with excess DBCO groups. We, then, metabolically glycoengineer MSCs and their secreted EVs through a ManNAz analog of ManNAc that replaces sialic acid membrane sugars with Azide groups, thus allowing to click EVs on DBCO-excess 2D hydrogels. Furthermore, the EVs are labeled with a DBCO-488 dye to allow fluorescent visualization during EV uptake. To overcome the challenge of the visualization of EVs under an epifluorescence microscope, due to their small size (~100nm), we use photo-expansion microcopy (photo-ExM), a method that physically enlarges our samples up to ~8x, thus allowing for high resolution images using conventional confocal microcopy. By varying the hydrogel substrate stiffness, the presented peptides (HAVDI, RGDS) and measuring cell membrane tension through fluorescence lifetime imaging (FLIM) probes, we uncover regulatory mechanisms between the cellular microenvironment and material properties and the endocytosis of EVs by MSCs.

In the second part of this study, we engineer granular microgel scaffold to direct MSC-derived EV protein and mi-RNA content towards and anti-inflammatory profile, through the utilization of anti-inflammatory peptides. Modified EVs are clicked on microgel scaffolds for the controlled in vivo delivery in a rat critical size calvarial defect model. After transplantation in a critical size calvarial defect of rats, we observed that macrophages infiltrating the EV laden microgel scaffolds polarized towards an M2a phenotype over 3 days, ultimately resolving inflammation over 7 days, significantly faster than the scaffolds without EVs. Ultimately, the granular microgels bio-degraded over 4-6 weeks, through the hydrolysis of their ester groups allowing for more efficient osteoanabolic activity.

Overall, we have utilized SPAAC hydrogels to address fundamental questions with respect to EV endocytosis, using state of the art techniques (photo-ExM, FLIM, -omics) to gain pivotal insight for the efficacy of EV therapeutics and subsequently engineered an innovative acellular system for the controlled delivery of anti-inflammatory EVs for in vivo bone regeneration.